Exam 3 Flashcards
Genome
All the DNA present in a cell or virus
Genotype
specific set of genes an organisms possesses
Major targets for antibiotics
- cell wall
- protein synthesis
- DNA/RNA replication
- tetrahydrofolic acid biosynthesis
Phenotype
collection of observable characteristics
Fred Griffith was able to observe what phenomenon?
transformation
Nucleoside
nitrogenous base & 5 C sugar
Nucleotide
nucleoside-phosphate
360 turn of the helix = ____ bases
10
Most RNA molecules are ____ stranded
single stranded
Double stranded RNA can be found in
viral genomes
Small regulatory RNA (sRNA)
- any RNA that is not making proteins
- not translated
- functional as RNA molecules
DNA replication is
semi-conservative
DNA polymerase
synthesizes complementary strand of DNA
T / F : DNA synthesis occurs only in one direction
T
New bases are only added to the ___ end of a DNA molecule
3’
What does DNA polymerase require?
- template
-primer - dNTPs
Template
directs synthesis of complementary strand
Primer
DNA or RNA strand
dNTPS
dATP, dTTP, dCTP, dGTP
Helicases
unwind DNA strands
Single stranded binding proteins (SSB)
keeps strands apart for replication to occur
DNA gyrase / topoisomerases
alter DNA supercoiling to relieve tension from rapid unwinding of double helix
Ligase
enzyme that attaches two pieces of single stranded DNA together
Leading strand is synthesized
continuously
Primase
synthesizes short complementary strands of RNA (~10 nucleotides) to serve as primers needed by DNA polymerase
Okazaki fragments
the short fragments of the lagging strand
DNA polymerase I
removes and replaces RNA primer with DNA
DNA ligase
forms a bond between 3’-hydroxyl of the growing strand and the 5’-phosphate of an Okazaki fragment
Exonuclease activity goes in what direction?
3’ to 5’
Catenates
form when the two circular daughter chromosomes do not separate
Okazaki fragments hinder
replication of ends of chromosomes
Coding strand
top strand
Template strand
- bottom strand
- read in 3’ to 5’ direction
Promoter is located ____ of the gene
upstream
Leader sequence is also known as
5’ untranslated region (5’ UTR)
Shine-Dalgarno
- ribosome binding site
-located within 5’ UTR - typically 6-10 nucleotides upstream of start codon
N-formylmehtionine
a modified amino acid used to initiate protein synthesis in bacteria
Stop codons
TAG, TAA, TGA, where coding region stops/translation stop site
T / F : no introns/exons in bacterial genes
T
Monocistronic
- encodes for one gene
- one gene = one protein
- eukaryotes
Polycistronic
- bacteria
- directions for >1 polypeptide contained within one mRNA molecule
2 subunits of RNA polymerase
core enzyme and sigma factor
Holoenzyme
core enzyme + sigma factor
Transcription is carried out by
RNA polymerase
Promoter
site where RNA polymerase binds to initiate transcription
- site that sigma factor binds to
T / F : transcription termination is at stop codons
F
Describe how Rho-independent transcription termination works
- no proteins involved
- DNA sequence alone results in termination
- RNA forms stem loop structure
- knocks RNA polymerase off the DNA
Describe how Rho-dependent transcription termination works
- Rho binds to mRNA at the rut site
- moves along mRNA in direction of RNA polymerase
- RNA polymerase pauses at Rho terminator
- Rho catches up and knocks RNA polymerase off
Final step in expression of protein encoding genes is
translation
T / F : double strands are not always antiparallel
F
Start codon
- start site for translation
- typically AUG
Sense codons
61 codons that specify amino acids
Code degeneracy
up to six different codons can code for the same amino acid
Codons UAA and UAG can code for
- stop
- amino acids
How many naturally occurring amino acids are there?
20
Rare amino acids encoded by some microbes
Selenocysteine (Sec, U, UGA), Pyrrolysine (Pyl, O, UAG)
If GUG and UUG are first
they are start codons and encode f-Met
N terminus
like 5’ end
C terminus
like 3’ end
Polyribosome
complex of mRNA with several ribosomes attached to it
T / F : transcription and translation cannot be coupled in bacteria and archaea
F
tRNA has terminal ____ sequence
CCA
Charged tRNA
tRNA with amino acid attached
tRNA without amino acid
uncharged
Aminoacyl-tRNA synthetases
catalyzes attachment of amino acid to tRNA
rRNA
- contributes to structure of ribosome
- 16S rRNA
- 23S rRNA
16S rRNA
- binds to Shine Dalgarno site on mRNA for protein synthesis initiation
- binds initiation factor 3 (protein needed for translation initiation) and 3’ end of charged tRNA
23S rRNA
ribozyme catalyzes peptide bond formation
What is usually removed after protein synthesis?
N-formylmethionine
Core enzyme
composed of 5 proteins and catalyzes RNA synthesis
Sigma Factor
has no catalytic activity but helps the core enzyme recognize the DNA at start of genes (promoter)
Transcription termination
occurs when core RNA polymerase dissociates from template DNA
- site where RNA synthesis ends
Translation
synthesis of polypeptide directed by sequence of nucleotides in mRNA (5’ to 3’)
Polyribosome
complex of mRNA with several ribosomes
Site of translation
ribosome
Two approaches to regulation
- regulation of gene expression
- alter activity of enzymes and proteins
Constitutive genes
housekeeping genes that are continuously expressed by the cell
Inducible genes
- genes that are typically off but can be switched on
- function in catabolic pathways
Repressible genes
- genes that are typically on but can be shut off
- repressible enzymes function in anabolic pathways
T / F: inducible enzymes are present only when their substrate is available
T
Inducer
effector molecule
Beta galactosidase hydrolyses ____ into ___ and ____
lactose, galactose and glucose
Beta galactosidase gene is only “on” when ____ is available
lactose
Typically, enzymes that function in biosynthetic pathways are products of
repressible genes
Induction and repression occur because of the activity of ____ containing ____
regulatory proteins, DNA binding domains
Regulatory proteins bind to DNA and can do one of what two things?
inhibit transcription or promote transcription
Repressors
inhibit transcription
Activators
promote transcription
Negative transcriptional control (overview and result)
- binding of regulatory protein (repressor) at DNA regulatory site (operator) inhibits initiation of transcription
- mRNA expression is reduced
_____ or _____ alter activity of repressor
inducers, corepressors
Positive control (overview and result)
- binding of a regulatory protein (activator) at a regulatory region on DNA (activator binding sites) promotes transcription initiation
- mRNA synthesis is increased
Inactive protein is activated by
inducer
Active protein is inactivated by
inhibitor
T / F: enzymes of a catabolic pathway are not only needed when the preferred substrate is available
F
3 structural genes coding for lactose uptake and metabolism
lacZ, lacY, lacA
Control of Lac operon is (+) or (-) ?
has negative and positive control
Expression of Lac operon is repressed by
Lacl repressor
____ of Lacl form and bind to three operator sites
tetramers
Allolactose binds ____
Lacl repressor
Catabolite activator protein
- regulates lac operon in response to presence or absence of glucose
When ___ is present, lac operon will not be activated
glucose
When ____ is absent, lac operon will be activated
glucose
Tryptophan operon consists of how many structural genes?
5
Trp operon only functions in the absence of ____
tryptophan
Attenuation
termination of transcription within the leader region
Trp experiences what type of regulation of transcription elongation?
attenuation
T/ F: transcription (attenuation) terminates after the gene is transcribed
F
Attenuation of the trp operon
- the trp leader sequence contains a short peptide called TrpL
- during transcription, the trp mRNA leader sequence forms secondary structure
- 2 hairpin loops form (regions 1 and 2, regions 3 and 4)
- regions 3 and 4 form transcription terminator
T/F: in attenuation of the trp operon, transcription and translation are coupled
T
TrpL has ___ Trp codons
2
What happens if amount of charged Trp tRNA is low?
ribosome stalls at region 1
Where does the anti-terminator form when there is a low amount of charged Trp tRNA?
2 and 3
What happens if amount of charged Trp tRNA is high?
ribosome does not stall at region 1
Where does the anti-terminator form when there is a high amount of charged Trp tRNA?
3 and 4
Riboswitches
a specialized form of transcription attenuation
Folding of ___ also known as _____ determines if transcription will continue/terminate
leader sequence, the riboswitch
Folding pattern altered in response to mRNA binding of a(n)
effector molecule
T / F: cis-acting regulatory elements are part of the same RNA transcript they regulate
T
Small RNAs (sRNAs)
- typically are complementary (antisense) to mRNA and function by base pairing
- may inhibit or enhance translation
- trans-acting RNAs
T/ F: sRNAs function as mRNA, tRNA, or rRNA
F
Why are global regulators important for bacteria?
because they must respond rapidly to wide variety of changing conditions
Regulon
set of genes or operons controlled by a common regulatory protein
Types of regulation global regulatory systems use
- regulatory proteins
- alternative sigma factors
- phosphorelay systems
- two component signal transduction systems
Many genes and operons are turned on or off in response to ____
environmental conditions
Common system used by bacteria to regulate gene expression in response to environmental conditions are _____
two-component signal transduction systems (TCSTS)
T / F: two component regulatory system is found in all three domains of life
T
Two proteins of the two component regulatory system
sensor kinase and response-regulatory protein
Sensor kinase (histidine kinase)
- extracellular receptor for sensing
- intracellular communication domain
Response-regulatory protein
- intracellular protein
- activated by sensor kinase
- DNA binding protein
^ activator, repressor
Activation of sensor kinase results in
phosphorylation of histidine
In RRP, phosphate group is transferred from ___ to ___
His (H), Asp (D)
EnvZ
phosphorylated in high osmolarity
OmpR
phosphorylated and regulates transcription
The OmpR/EnvZ two component signal transduction system
regulates expression of outer membrane proteins (OmpC and OmpF) depending on osmolarity
OmpC
makes smaller pore in membrane, lower levels of diffusion into the cell
OmpF
makes larger pore in membrane, allows more diffusion of solutes
High osmolarity leads to
autophosphorylation of EnvZ —> –> –> OmpR activates OmpC
Low osmolarity leads to
no phosphorylation of EnvZ, no activation of OmpC
T / F: machinery involved in replicating and expressing archaeal genomes is more like that of eukaryotes than that of bacteria
T
DNA polymerase catalyzes synthesis of
complementary strand of DNA
Template
directs synthesis of complementary strand
Primer
DNA or RNA strand
dNTPs
dATP, dTTP, dCTP, dGTP
Eukaryotic chromosomes are ____ while bacterial are ___
linear, circular
Polymerase enzymes require
- template
- primer
- dNTPs
Bacterial chromosome is ___ Mb
2.5
Eukaryotic chromosomes are wound around
histone proteins
3 DNA polymerases responsible for eukaryotic DNA replication
- α-primase
- Pol ε
- Pol δ
Pol δ
appears to synthesize the lagging strand
Pol ε
appears to synthesize the leading strand
α-primase
lays down an RNA/DNA hybrid starting point
Chromosome ends may be digested by
DNAses
What are some challenges presented by linear DNA molecules?
- chromosome ends may be digested
- DNA ends can fuse with other DNA molecules
- the “end replication problem”
Telomerase
ribonucleoprotein complex enzyme that forms the telomeres and maintains them
Telomerase has an internal _______ that partially matches the _______
RNA template, G-tail sequence
Telomerase uses the _____ of the G tail as a DNA synthesis starting point
3’ OH
The internal RNA sequence is used as the template for
DNA synthesis
The _______ activity of telomerase increases the length of the G-tail
reverse transcriptase
Lengthening the G-tail creates enough room to make a(n) ____ on the _____
RNA primer, lagging strand
T / F: archaeal chromosomes are more similar to eukaryotes than bacteria
F
T / F: some archaea have histones associated with their chromosomes
T
Archaeal chromosomes, like bacterial, are ___
circular
Archaeal and bacterial chromosomes are similar in ____ and ___
size, structure (circular)
Most archaea have how many origin of replication?
one
_________ in archaea are similar to those of eukaryotes
replisome proteins
___ family use the same family of DNS polymerases as eukaryotes
B
T / F: on a molecular level, transcription is very similar in all organisms
T
What direction is RNA produced in?
5’ to 3’
Promoter is located at the ____
start of the gene
Two characteristics of a promoter
- recognition/binding site for RNA polymerase
- orients polymerase
The bacterial RNA polymerase is known as
holoenzyme
Holoenzyme is composed of
sigma factor, core enzyme
Sigma factor
has no catalytic activity but helps the core enzyme recognize the start of genes
How many polymerase do bacteria have for transcription?
1, have multiple sigma factors
Where does eukaryotic transcription occur?
nucleus
Exons
regions coding for protein that end up in the mRNA
Introns
code for RNA that is never translated into protein
T / F: most eukaryotes have singlet protein-coding genes (monocistronic transcripts)
T
3 major types of RNA polymerase
RNA pol II
RNA pol I
RNA pol III
RNA polymerase II catalyzes
mRNA synthesis
RNA polymerase I catalyzes
rRNA synthesis
RNA polymerase III catalyzes
tRNA synthesis
T / F: promoters do not differ between eukaryotes and bacteria
F
Elements that define the core promoter
- TATA box
- BRE (TFIB recognition element)
- Inr (initiator element)
- DPE (downstream promoter element)
T / F: eukaryotic and archaeal promoters share sequences
T
T / F: no sigma factor in eukaryotes or archaea
T
Eukaryotes and archaea use _____ instead of sigma factors
transcription factors
Transcription factors
bind to DNA and line the polymerase up to it correctly
How must initial transcripts be modified before they are ready to be translated?
- 5’ cap of 7-methylguanosine added
- removal of introns/splicing together exons
- addition of 3’ poly a tail
Spliceosome
large complex of proteins and RNA molecules unique to eukaryotes, removes introns
Alternative splicing
different ways of joining exons together
T / F: alternative splicing can be found in any domain
F, only in eukaryotes
What mechanism allows for a smaller number of genes to code for a greater number of products in cells?
alternative splicing
Similarities between archaeal transcription and eukaryotic
- RNA polymerase resembles polymerase II
- archaeal promoters and binding of the RNA polymerase
- no sigma factors
Similarities between archaeal transcription and bacterial
- transcription occurs in the cytoplasm
- coupling of transcription/translation
- mRNA is polycistronic
- introns are rare
Introns in archaea and bacteria are thought to be excised by
different methods, maybe something like tRNA splicing in eukaryotes
Eukaryotic translation requires more or less initiation factors for proper positioning on the mRNA?
more
Eukaryotic ribosome size:
Bacterial ribosome size:
80S
70S
Eukaryotic initiation factors bind to
the 5’ cap
PABPs bind to
3’ poly-A tail
The cap/tail protein complexes form a _____, activating the _____
bridge, mRNA molecule
Initiator tRNA (Met) interacts with ____ to form ____
40S ribosomal subunit, 43S subunit
43S binds to the activated ______
(bridged) mRNA
____ is recruited after _____ to begin elongation steps
60S, 43S unit scans the mRNA for start codon
T / F: elongation and termination are similar in eukaryotes and bacteria
T
Polysomes
observed in eukarya, more than one ribosome translating the same mRNA molecule
T / F: no N-formylmethionine in archaeal translation
T
Transcription and translation are ____ in archaea
coupled
Polycistronic transcripts
each coding region translated into separate protein
Regulation of cellular processes can take place at what 3 levels
transcriptional, translational, posttranslational
Activator binding sites include
upstream activating sequences, and enhancers
Upstream activating sequences (UASs)
near the promoter of the gene regulated
Enhancers
- either upstream or downstream of the promoter
- exert effects from a distance
Repressor binding sites
silencers
Histones act as a _____ in transcription
road block
Acetylation of histones _____ transcription
promotes
Methylation of histones _____ transcription
represses
T / F: histone acetylation is a gene regulation/control mechanism unique to eukaryotes
T
Examples of regulatory mechanisms for gene expression
antisense RNAs that function at the translation level, riboswitches, sRNA molecules
Riboswitches
- in bacteria: transcription elongation/translation
- control whether or not RNA splicing occurs
sRNA molecules
regulate gene expression via alternative splicing modulation
Other RNA based regulatory mechanisms in eukaryotes
- micro RNAs (miRNAs)
- small interfering RNAs (siRNAs)
siRNAs
- form complexes with proteins including argonaute (AGO)
- function at translational level
- prevent translation initiation and elongation
- can also degrade mRNAs
Levels of gene regulation
- transcription initiation
- splicing efficiency
- mRNA trafficking
- mRNA stability/degradation
- translational rate
- post-translational modification
T / F: regulation of transcription initiation in archaea has similar machinery to eukaryotes
T
Mutations
stable, heritable changes in sequence of bases in DNA
Most common kind of mutation:
point
Point mutations
- single nucleotide polymorphism, single nucleotide is changed (A to G)
- insertion or deletion (AAAA to AAA or AAA to AAAA)
consequence of mutation depends on ___ and ___
type and location
If a mutation occurs within a gene, it is called a(n)
coding DNA sequence, CDS, mutation
If a mutation is between two genes, it is called a(n)
intergenic (IG) mutation
An SNP CDS mutation would result in what?
a silent mutation, no amino acid change OR missense, amino acid change
An SNP IG mutation would result in what?
- no effect
- alter gene expression
- sRNAs
An Indel CDS mutation would result in what?
a frameshift, multiple amino acid changes/stop codon
An Indel IG mutation would result in what?
- no effect
- alter gene expression
- sRNAs
Consequences of mutations manifest as
phenotypic changes
What are consequences of a change in protein sequence?
- loss of function
- gain of function
Error rate of DNA polymerase
10E-9 errors per base
Errors per genome replication (bacteria)
5Mbp
Mismatch repair
- type of excision repair
- mismatch correction enzyme (MutS) scans newly synthesized DNA for mismatched pairs
- mismatched pairs removed and replaced by DNA pol and DNA ligase
- repair system uses methylation to “know” which strand is correct
DNA methylation
used by E. coli mismatch repair system to distinguish old DNA strands from new DNA strands
The E. coli mismatch repair system cuts out the mismatch from the ____ strand
unmethylated
Vertical gene transfer
transfer of genes from parents to progeny
Genetic variation in VGT is primarily due to
- fusion of male and female gametes
- crossing over between sister chromatids during meiosis
Source of genetic variability for eukaryotes
sexual reproduction
Genetic variability (in prokaryotes) primarily due to
horizontal gene transfer
HGT differs from VGT in what ways?
- transfer of genes from one independent, mature organism to another
- genes can be transferred to the same or different species
- if transfer is stable, recipient acquires function of transferred gene
What does it mean for DNA to be stable?
self replicating, able to be integrated onto chromosome
Integrating into the host genome occurs by
recombination
2 major types of recombination
homologous recombination, site specific recombination
What is the most common type of recombination?
homologous
Homologous recombination
- occurs between two long sequences of DNA with same/similar sequence
- double-strand break occurs
- adjacent molecules are rejoined to each other
- involves the RecA protein
Site-specific recombination is important in
insertion of viral genome into host chromosomes and transposons
3 important differences between site-specific recombination and homologous
- does not require long regions of DNA homology
- recombination occurs at specific target sites in DNA molecules
- mediated by specific enzymes called recombinases
2 major types of transmissible DNA
- transposable elements
- plasmids
3 mechanisms for HGT
- conjugation
- transformation
- transduction
Transposition
process by which small segments of DNA move about the genome
Insertion sequences
simplest transposable elements
Composite transposons (Tn)
transposable elements which contain “extra” genes, flanked by two IS
Unit transposons are between ___ and ___
IS, composite Tn
Unit transposons
like expanded IS but can contain “extra” genes
Inverted repeats
- short stretch of identical nucleotides in opposite orientation
- marks the ends of the IS/Tn
Transposase
enzyme responsible for site specific transposition
Direct repeats
- host DNA that is the site for insertion
- duplicated during insertion
2 mechanisms of action for transposition
- simple transposition (cut and paste)
- replicative transposition (copy and paste)
Simple transposition
- cut and paste
- transposase catalyzes excision of TE
- TE is bound by transposase and migrates to new insertion site
- new target site is cleaved and TE ligated into site
- target site is replicated in the process (DR)
Replicative transposition
- original transposon remains at parental site in DNA
- copy is inserted in target DNA
- retrotransposons
Plasmids
- small, extrachromosomal circular DNA molecules
- exist and replicate independently of chromosome
- contain non essential genes
Episomes
can integrate reversibly into the host chromosome
Conjugative plasmids
can transfer copies of themselves to other bacteria during conjugation
Retrotransposons
- RNA copy of TE is made
- reverse transcribed into DNA
- integrates at new site
Plasmid is replicated by
rolling circle method
F factor codes for
sex pilus and all machinery needed for plasmid transfer
F plasmid is an
episome
Integration of F plasmid occurs at
insertion sequences
When F factor is not integrated into chromosome, cell is called ____
F+
When F factor is integrated into chromosome, cell is called ____
HFr
T / F: conjugation cannot occur between HFr and F- strains
F
F’ plasmid forms when
the F factor incorrectly excises from the host chromosome
T / F: in F’ conjugation, some of the F factor is left behind in the host chromosome and/or some host genes have been removed along with some of the F factor
T
Transformation
uptake of extracellular DNA by a bacterial cell
Competent cells
bacterial cells that have the ability to take up DNA
Competent cells are ____ when they take up DNA
transformed
DNA being transformed may be ___ or ___
circular (plasmid) or linear (fragment of genomic DNA)
For linear DNA to be stably transformed, it must
integrate into the chromosome
Transduction
the transfer of bacterial genes by viruses
Lytic
host cell is destroyed
Lysogenic
viral DNA integrates into the host genome (becoming prophage)
Two potential cycles of transduction
lytic and lysogenic
Phage mediated transduction
- host genes transferred by bacteriophage
- typically an “accidental” process
- results from errors in phage life cycle
2 types of phage mediated transduction
- generalized transduction
- specialized transduction
Generalized transduction
- occurs because of errors in lytic cycle
- any part of bacterial genome can be transferred
- during viral assembly, fragments of host DNA mistakenly packaged into phage head
Specialized transduction
- occurs because of errors in lysogenic cycle
- occurs when prophage is incorrectly excised
- new virions contain part of the viral genome plus host DNA surrounding viral integration site
- host DNA is introduced into recipient cell
- can recombine into genome or integrate with partial phage genome
- host DNA transferred is that surrounding viral integration site
Generalized transducing particles
- bacterial DNA wrapped in viral proteins
- no viral DNA
- can be easily transferred to another bacterial cell
Phage lambda specialized transduction
recipient becomes gal+
Mechanisms of drug resistance
- modification of target enzyme or organelle
- inactivation of drug
- efflux pumps
- use of alternative pathways or proteins
How does antibiotic resistance occur?
- mutations (arise spontaneously and are then selected for)
- horizontal gene transfer of resistance genes
Resistance genes can be found on
- bacterial chromosomes
- plasmids
- transposons
Genetic engineering
deliberate modification of organism’s genetic information by directly changing the sequence of nucleic acids in its genome
Recombinant DNA
artificially created DNA sequences, results from combining 2 strands of DNA together
Cloning
generating a large number of genetically identical DNA molecules
Biotechnology
use of biological organisms to form useful products
Industrial microbiology
use of microbes to manufacture important compounds
What are the 2 main reasons you might want to express a foreign gene in a host cell?
- to determine its function
- to purify the protein
Recombinant DNA technologies
- restriction enzymes
- genetic cloning
- PCR
-DNA sequencing
Restriction enzymes (RE)
- recognize and bind specific sequences in DNA called recognition sites
Type II RE cleave
DNA at/around the site
Type I and III cleave
a defined distance from this site
Cleavage with restriction enzymes may produce
blunt or sticky ends
Palindromes
same going forward as it is going backwards
Vectors
carriers of foreign DNA
4 types of cloning vectors
- phages and viruses
- cosmids
- artificial chromosomes
- plasmids (most commonly used)
Cloning a gene
- foreign DNA combined with/inserted into a cloning vector
- newly created plasmid is a recombinant DNA molecule
- inside a new host cell plasmid vector replicates
- maintains foreign DNA fragment (gene)
Characteristics of good cloning vectors
- replicate autonomously
- easy to purify
Requirements for vectors
- origin of replication
- selectable marker (ex: antibiotic resistance gene)
Multiple cloning site (MCS)
- site where cloned gene to be inserted into the plasmid vector
- contains many unique RE sites
- may contain selection gene
PCR
technique that enables DNA amplification
Specificity of PCR arises from
DNA primers
Oligonucleotides
short DNA molecules that flank the DNA sequence being amplified, serve as primers for DNA polymerase
PCR cycle
- DNA is denatured
- primers anneal to target DNA
- target DNA is synthesized (amplified)
- repeat 34x
PCR reaction mix contains:
- primers
- template DNA (target)
- thermostable DNA polymerase (taq)
- dNTPs (dATP, dCTP, dTTP, dGTP)
Number of copies of DNA produced in PCR reaction =
2^n, where n = how many cycles were done
Uses of PCR
- simplifies gene cloning
- generates DNA fragments
- may amplify environmental genes without culturing the microbes
- diagnostic purposes
Limitations of PCR
- mutations due to Taq polymerase
- cannot have a completely unknown sample
Most common method for determining DNA sequences
Sanger DNA sequencing
How does Sanger sequencing work?
referred to as the chain-termination DNA sequencing method, uses dideoxynucleoside triphosphates (ddNTP), strand synthesis terminates when ddNTP is incorporated
Sanger sequencing original method
newly synthesized DNA strands were labeled with radioactive atom, electrophoresed and detected with X ray film
Sanger sequencing, modern method
automated sequencing, uses four different fluorescent color dyes instead of radio-labeled ddNTP, electrophoresis and laser beam determines order
NGS (next generation DNA sequencing)
sequences millions of DNA strands simultaneously
Sanger vs NGS (bp comparison)
Sanger: 1,000 bp/reaction
NGS: 10-100 billion bp/reaction
